Understanding the organization of chromosomes into functional domains that operate in regulating transcription and other cellular processes is the long term goal of this proposal. Sequences that are thought to specify the borders of transcriptional domains are known as boundary sequences. Two different boundary sequences, the scs sequence from Drosophila and the ROR from the Xenopus rRNA genes, recreate transcriptional domains on plasmids injected into Xenopus oocytes. Development of this assay has shown that chromosomal context is not required for boundary sequence function, and that the role of boundary sequences is likely to be conserved from insects to vertebrates. The plasmid-based oocyte injection assay is a significant advance in the study of boundary sequences because it enables us to test the mechanism of action of boundaries without the constraints imposed by chromosomal integration assays. The aims of this proposal are to learn more about the function of these sequences using varied DNA templates in the oocyte injection assay, define the crucial sequence elements for scs and ROR activity, and to use this information to begin the biochemical characterization and purification of the proteins that mediate boundary sequence function.